As levels of antibiotic resistance in pathogens, infections by multi-drug resistant strains, and incidence of cancer continues to increase worldwide, new antibiotics are urgently needed to combat these growing threats to human health. Unfortunately, only few biopharmaceutical companies have retained aggressive natural product discovery programs due to the continued decline in promising candidates from traditional screening methods and low levels of projected profits. This project seeks to answer the challenge for new antibiotics through the discovery of novel nitroaromatic natural products from cultivated actinomycetes and the characterization of their biosynthesis. These compounds are an understudied class of natural products and a promising source of new drugs and medicines. Although only a small number of nitroaromatic natural products have been reported to date, the wide-ranging bioactivities and a rich history as medicines of these compounds has served as inspiration for the synthesis of new drugs with this functional group, which continues even today. A genome-mining approach will be used to screen the entire USDA-Agricultural Research Service (ARS) actinomycete strain collection (ca 7,500) for the discovery of novel nitroaromatic antibiotics. Genomic DNA from each strain in the entire USDA-ARS actinomycete collection will be extracted and arrayed in 96-well format, creating a unified high-throughput platform for natural product discovery. Strains with the ability to synthesize nitroaromatic compounds will be identified using PCR screening using degenerate primers to aromatic amine dioxygenases, a unique group of enzymes responsible for the addition of the nitro group onto aromatic rings, and bioinformatic analyses of sequenced genomes for natural product gene clusters containing nitric oxide synthase. Media extracts from candidate strains will be screened by a variety of chromatography, derivatization, and mass spectrometry analyses to identify novel and bioactive nitroarenes. Select compounds will be purified (prioritized based on their abundance and preliminary bioactivities) for characterization of their chemical and biological properties. Simultaneously, recent advances in next-generation sequencing and molecular genetics will be applied to uncover the basis for their biosynthesis. Whole genome sequencing, gene-knockouts, and heterologous expression experiments will be used identify and elucidate the role of pathway genes in nitroarene biosynthesis. Results from these studies are expected to not only yield new compounds that may useful as drugs and antibiotics, but also expand our knowledge about this relatively unexplored group of natural products. PUBLIC HEALTH RELEVANCE: As levels of antibiotic resistance in pathogens, infections by multi-drug resistant strains, and the incidence of cancer continues to increase worldwide, new medicines are urgently needed to combat these growing threats to human health. This project seeks to answer this challenge by discovering new drugs and antibiotics produced from microorganisms and investigating how they are made.